Recent progress in stem cell research suggests that an unlimited source of beta-cells can be generated in vitro from cultures of stem cells. This promising approach is hampered by the limited knowledge about the specification of pancreatic endoderm and its specialized cell-types. Furthermore, the scarcity of research tools obstructs efforts to elucidate the molecular signals that control these processes. In this proposal we will investigate the role of Nkx6 family genes in pancreatic specification and beta-cell development. We will analyze mice deficient for Nkx6.1 as well as double mutant mice. Additionally, we will perform gain-of- function studies using the newly described chicken in ovo endoderm electroporation to study the role of Nkx6 family genes in the specification of pancreatic endoderm. We will also generate a set of mouse lines genetically manipulated to express Blue, Green, and Red fluorescent proteins under control of the regulatory sequences of the Pdx1, neurogenin3, and Nkx6.1 genes, respectively. These animals will provide a valuable resource from which stem cells can be derived. Specifically, Projects 2, 3, and 4 in the overall application will derive early pancreatic progenitor cells, ES-cell s, and bone marrow stem cells, respectively. The investigators in these projects will then be able to monitor expression of these key transcription factors non-invasively and also be able to FACS sort specific progenitor populations using these genetic tags. In this proposal we will additionally employ these mice to generate cDNA from early pancreatic progenitor cells FACS sorted after their expression of Pdx1, neurogenin3, and Nkx6.1 and subject this to micro- array analyses to define the transcriptome of the various progenitor populations i.e. endocrine versus exocrine progenitor cells. Lastly, we will search for proteins capable of physically interacting with neurogenin3 and Nkx6.1 using two-hybrid experiments in order to search for "novel" genes involved in pancreas development.